Epstein-Barr Virus (EBV) is associated with the development of several lymphomas and carcinomas, and HCMV infection has recently been associated with human glioblastoma. During the previous funding period we developed an approach to define EBV genes that act as drivers for Burkitt's Lymphoma (BL) by forcing the loss of the viral genome from recently isolated BL cells. We discovered that constitutive expression of EBV's BART miRNAs in canonical, as well as Wp-restricted, BL cells prior to loss of the viral genome rescues them from apoptosis and reduced proliferation, respectively. We also identified cell mRNAs regulated by the BART miRNAs and found both IP07 and Caspase 3 to be among their targets. These findings indicate that EBV's BART miRNAs are drivers of lymphomas as measured in cell culture. We shall now extend these findings to in vivo measurements in NSG mice, and will use a new humanized mouse model (developed during the previous funding period) to study the roles of the BART miRNAs on EBV-induced lymphomas in the context of a human immune system. In the last funding period we also showed that Hsp90 inhibitors decrease EBNA1 expression and repress EBV-induced lymphoproliferative disease in SCID mice. Our new preliminary results show that Hsp90 directly interacts with both the EBV- and HCMV- encoded kinases (BGLF4 and UL97, respectively), and that Hsp90 inhibitors greatly reduce expression of both BGLF4 and UL97, proteins required for efficient productive infections. We shall now extend these studies to examine the use of these drugs for treatment of de novo lytic and reactivating EBV and HCMV infections both in vitro and in the humanized mouse model. We hypothesize that 1) viral miRNAs contribute to the survival and growth of BLs; 2) viral microRNAs promote efficient establishment of viral latency and help EBV to evade the host- immune response; and 3) Hsp90 inhibitors can be used to treat the lytic forms of EBV and HCMV at least partially through their effect on the essential virally-encoded protein kinases. Our Specific Aims are 1) to ex- amine the roles of BART miRNAs in BLs in NSG xenograft models; 2) to examine the roles of BART miRNAs on viral pathogenesis, lymphoma formation and viral reactivation in humanized mice, and 3) to determine if Hsp90 inhibitors can be used to treat the lytic forms of EBV and HCMV infection in vitro and in humanized mice. The proposed studies will involve extensive interactions with Dr. Mertz in Project 4 (examining the ef- fect of the BARTs on EBV reactivation) and Dr. Lambert in Project 1 (who will provicde expertise in NSG xen- ograft models, and in preparing paraffin-fixed slides of EBV-infected tissues). RELEVANCE (See instructions): Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) infection are associated with B cell lympho- mas and glioblastomas, respectively, and HCMV causes life-threatening illness in cancer patients. Better animal models are needed to understand how EBV promotes lymphoma, and to develop and test new thera- pies for EBV as well as HCMV. We will use a new humanized mouse model, as well as xenograft animal models, to dissect the roles of the EBV-encoded BART miRNAs in EBV-induced lymphomas, and to test whether Hsp90 inhibitor drugs can be used to treat both EBV and HCMV at non-toxic doses.